Latest revision as of 13:38, 20 October 2021

T7-RBS-His-DcAFP-Term

DcAFP

DcAFP is a protein coding region that codes for an antifreeze protein (AFP). AFPs can bind to ice crystals and thereby prevent further ice growth. They are produced by organisms to survive in extremely cold environments. Activities of AFPs can be characterized by their thermal hysteresis (TH) or by their ice recrystallization inhibition (IRI). TH activity corresponds to the lowering of the freezing point without changing the melting point of a solution. IRI activity inhibits the growth of large ice crystals at the expense of smaller ones. The combination of these activities, which vary depending on the protein structure, prevents the freezing of body fluids and cell damage in organisms that live in environments with extremely cold temperatures.

Profile

Usage and Biology

The DcAFP protein coding region was used in the following composite parts (add links). It was expressed in E. coli strain BL21 (DE3) and purified. Various tests and assays were performed to characterize and verify the functionality of this ice-binding protein. DcAFP has low TH and high IRI activity, and it can bind to ice crystals and inhibit their growth. The aim of our project was to use it on its own or in a mixture of other antifreeze proteins to develop a solution which could be applied on sensitive plant tissues and thereby protect crops from frost damage.

DcAFP is naturally produced by Daucus carota. Its TH activity is around 0.35K at 1.0mg/mL^[1].

Antifreeze proteins bind to ice crystals and inhibit their growth. Ice crystals don’t have the same molecular arrangement on each of its faces and certain AFPs tend to adsorb to the basal plane of the crystals whilst others tend to adsorb to the prism plane. Hyperactive AFPs can bind to every plane^[2]. The mechanism by which AFPs suppress the freezing point of a solution still isn’t completely understood. Nevertheless, AFPs are believed to attach irreversibly to the plane of a growing ice crystal. As the protein adsorbs to the growing ice plane, growth at the site is suppressed, producing bulges in between the adsorbed proteins^[3]. Due to the Gibbs-Thomson effect, the freezing point will be depressed^[4].

Characterization

To reduce frost damage during late spring freeze, we wanted to develop a solution containing antifreeze proteins, which bind to ice crystals and inhibit their growth. DcAFP was therefore chosen as one of three AFPs and cloned and expressed in E. coli BL21 (DE3).
Two different vectors were used for cloning: pET-17b and pColdI, containing a T7 promoter and a cold-shock protein A (cspA) promoter respectively.

Cloning

Figure 1: (a) DcAFP in the DcAFP-pColdI construct was visualized by a 1% Agarose Gel. Left to right: L - 1 kb DNA Ladder, 1 – pCold-I-DcAFP (588). (b) DcAFP in the pET-17b construct was visualized by a 1% Agarose Gel. Left to right: L - 1 kb DNA Ladder, 2 – pET-17b-DcAFP (640 bp).

DcAFP was cloned into the pET-17b plasmid using restriction and ligation via HindIII and XhoI and Gibson Assembly. The vector includes an ampicillin resistance gene and a lac operator, which allows for IPTG induced expression. The final construct was checked with a 1% Agarose gel (Fig.1) and via sequencing.

DcAFP was cloned into the pColdI plasmid using restriction and ligation via NdeI and XhoI and Gibson Assembly. The vector includes an ampicillin resistance gene and a lac operator, which allows for IPTG induced expression. The final construct was checked with a 1% Agarose gel (Fig.1) and via sequencing.

Sequence and Features

References

1. ↑ Worrall D, Elias L, Ashford D, Smallwood M, Sidebottom C, Lillford P, et al. A carrot leucine-rich-repeat protein that inhibits ice recrystallization. Science (80- ) [Internet]. 1998 Oct 2 [cited 2021 Oct 8];282(5386):115–7. Available from: https://www.science.org
2. ↑ Olijve LLC, Meister K, DeVries AL, Duman JG, Guo S, Bakker HJ, et al. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins. Proc Natl Acad Sci [Internet]. 2016 Apr 5 [cited 2021 Oct 10];113(14):3740–5. Available from: https://www.pnas.org/content/113/14/3740
3. ↑ LM S, AV T. Kinetic pinning and biological antifreezes. Phys Rev Lett [Internet]. 2004 Sep 17 [cited 2021 Oct 10];93(12). Available from: https://pubmed.ncbi.nlm.nih.gov/15447309/
4. ↑ Pereyra RG, Szleifer I, Carignano MA. Temperature dependence of ice critical nucleus size. J Chem Phys [Internet]. 2011 Jul 21 [cited 2021 Oct 10];135(3):034508. Available from: https://aip.scitation.org/doi/abs/10.1063/1.3613672